1. Field of the Invention
The present invention is directed to liquid and lyophilized formulations of Keratinocyte Growth Factor-2 (KGF-2) and derivatives thereof. This invention further relates to formulations of KGF-2, especially topical and injectable formulations, that can be employed for therapeutic use in indications requiring soft-tissue growth and regeneration.
2. Related Art
The fibroblast growth factor family has emerged as a large family of growth factors involved in soft-tissue growth and regeneration. It presently includes several members that share a varying degree of homology at the protein level, and that, with one exception, appear to have a similar broad mitogenic spectrum, i.e., they promote the proliferation of a variety of cells of mesodermal and neuroectodermal origin and/or promote angiogenesis.
KGF was originally identified as a member of the FGF family by sequence homology or factor purification and cloning. Keratinocyte growth factor (KGF) was isolated as a mitogen from a cultured murine keratinocyte line (Rubin, J. S. et al., Proc. Natl. Acad. Sci. USA 86:802-806 (1989)). Unlike the other members of the FGF family, it has little activity on mesenchyme-derived cells but stimulates the growth of epithelial cells. Keratinocyte growth factor is produced by fibroblasts derived from skin and fetal lung (Rubin et al. (1989)). The Keratinocyte growth factor mRNA was found to be expressed in adult kidney, colon and ilium, but not in brain or lung (Finch, P. W. et al. Science 245:752-755 (1989)). KGF displays the conserved regions within the FGF protein family. KGF binds to the FGF-2 receptor with high affinity.
Impaired wound healing is a significant source of morbidity and may result in such complications as dehiscence, anastomotic breakdown and, non-healing wounds. In the normal individual, wound healing is achieved uncomplicated. In contrast, impaired healing is associated with several conditions such as diabetes, infection, immunosuppression, obesity and malnutrition (Cruse, P. J. and Foord, R., Arch. Surg. 107:206 (1973); Schrock, T. R. et al., Ann. Surg. 177:513 (1973); Poole, G. U., Jr., Surgery 97:631 (1985); Irvin, G. L. et al., Am. Surg. 51:418 (1985)).
Wound repair is the result of complex interactions and biologic processes. Three phases have been described in normal wound healing: acute inflammatory phase, extracellular matrix and collagen synthesis, and remodeling (Peacock, E. E., Jr., Wound Repair, 2nd edition, W B Saunders, Philadelphia (1984)). The process involves the interaction of keratinocytes, fibroblasts and inflammatory cells at the wound site.
It is desirable to formulate polypeptides that are capable of promoting and enhancing soft-tissue growth and regeneration in pharmaceutical compositions that (1) are stable over prolonged periods of storage, (2) increase the pharmacological activity or effectiveness of the the polypeptide and/or (3) allow facile application or administration of the polypeptide in therapeutic regimens.
The present invention is directed to liquid and lyophilized formulations of KGF-2 and deletion or point or substitution mutants thereof (referred to herein as KGF-2 polypeptides). This invention further relates to the use of such formulations of KGF-2 polypeptides to promote or accelerate soft tissue growth or regeneration, for example in wound healing, or in treating mucocytis or inflammatory bowel disease. Preferred formulations of the present invention employ novel mutant forms of KGF-2, and in one embodiment employ a deletion mutant referred to herein as KGF2-xcex9433. The co-ingredients employed in the formulations (1) provide storage stability to the KGF-2 polypeptide, (2) further enhance soft-tissue healing activity of the therapeutic composition, and/or (3) provide the KGF-2 polypeptide in an active form while allowing facile application and administration for particular therapeutic purposes.
A first aspect of the present invention relates to a formulation comprising a KGF-2 polypeptide and a buffering agent having a buffering capacity of between about pH 5.0 and about pH 8.0. Useful buffers include phosphate, acetate, aconitate, succinate, malate, carbonate and citrate buffers, citrate being preferred.
A second aspect of the invention relates to a formulation comprising a KGF-2 polypeptide, a lyophilization bulking agent and a buffering agent having a buffering capacity of between about pH 5.0 and about pH 8.0. Useful buffers include phosphate, aconitate, succinate, malate, carbonate and citrate buffers, citrate being preferred.
A third aspect of the invention relates to a formulation comprising a KGF-2 polypeptide and a thiol-containing compound, preferably monothioglycerol, capable of stabilizing the KGF-2 polypeptide. This formulation preferably includes a buffering agent having a buffering capacity of between about pH 5.0 and about pH 8.0. This formulation may also include one or more antioxidants and or one or more metal chelating agents.
A fourth aspect of the present invention relates to a formulation comprising a KGF-2 polypeptide, a buffer, and a high molecular weight compound that causes the formulation to gel at a certain predefined temperature. A preferred high molecular weight compound is a Pluronic or Poloxamer polyoxyethylene-polyoxypropylene block copolymer. A thiol-containing compound, such as monothioglycerol, can be included in the formulation to provide added stability to the polypeptide.
A fifth aspect of the present invention relates to a formulation comprising a KGF-2 polypeptide, a buffering agent and a thickening agent. Thickening agents are used to increase the viscosity of the formulation. Preferred thickening agents are carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), hydroxypropylmethyl cellulose (HPMC), Natrosol, and Carbomers.
In addition, the formulations of the present invention may also include metal chelating agents, antioxidants or thiol-containing compounds, such as ascorbic acid ester, monothioglycerol, cystein, tocopherols, butylated hydroxyanisole, sodium sulphate, sodium bisulfite, and sodium metasulfite and preservatives such as phenol, chlorobutane, benzylalcohol, methyl parabens and propyl parabens. The formulations of the present invention may also have an nitrogen blanket overlay on the head space of the vial. Additionally, the formulations of the present invention may be include purging the formulation buffer with helium, argon, or nitrogen.